EP-4737565-A1 - UTILIZATION OF SUCROSE FOR INCREASED PRODUCTION OF TERPENES IN RHODOBACTER MICROBIAL CELL FACTORIES

Abstract

The present invention relates to a Rhodobacter cell engineered to utilize sucrose, comprising: (i) a heterologous enzyme having invertase activity; (ii) a heterologous enzyme having sucrose permease activity; and, optionally, (iii) a heterologous enzyme having fructokinase activity, and to a method for producing a Rhodobacter cell engineered to utilize sucrose. The invention further relates to a method for preparing a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a flavour, an aroma compound, an UV scavenger, a natural colorant, a natural crop protectant or an isoprenoid moiety containing compound such as coenzyme Q10, comprising culturing the Rhodobacter cell according to the invention in a culture medium comprising sucrose as carbon source for the monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene, flavour, aroma compound, UV scavenger, natural colorant, natural crop protectant or the isoprenoid moiety containing compound such as coenzyme Q10. In addition, the invention pertains to the use of the Rhodobacter cell of the invention for the production of monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, flavours, aroma compounds, UV scavengers, natural colorants, natural crop protectants or for production of isoprenoid moiety containing compounds such as coenzyme Q10.

Inventors

• CANKAR, Katarina
• VAN HOUWELINGEN, Adele Margaretha Maria Liduina
• BOSCH, HENDRIK JAN
• BEEKWILDER, MARTINUS JULIUS

Assignees

• Isobionics B.V.
• Stichting Wageningen Research

Dates

Publication Date

20260506

Application Date

20241031

Claims (18)

1. A Rhodobacter cell engineered to utilize sucrose, comprising: (i) a heterologous enzyme having invertase activity; (ii) a heterologous enzyme having sucrose permease activity; and, optionally, (iii) a heterologous enzyme having fructokinase activity.
2. The Rhodobacter cell of claim 1, comprising: (i) a heterologous enzyme having invertase activity which comprises or consists of an amino acid sequence as shown in SEQ ID NO. 2, or an amino acid sequence having at least 70% sequence identity to SEQ ID No. 2; (ii) a heterologous enzyme having sucrose permease activity which comprises or consists of an amino acid sequence as shown in SEQ ID NO. 6, or an amino acid sequence having at least 70% sequence identity to SEQ ID No. 6; and, optionally, (iii) a heterologous enzyme having fructokinase activity which comprises or consists of an amino acid sequence as shown in SEQ ID NO. 4, or an amino acid sequence having at least 70% sequence identity to SEQ ID No. 4.
3. The Rhodobacter cell of claim 1 or 2, comprising: (i) a heterologous nucleic acid sequence as shown in SEQ ID No. 1, or a heterologous nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 1, wherein said heterologous nucleic acid sequence encodes an enzyme having invertase activity of SEQ ID No. 2; (ii) a heterologous nucleic acid sequence as shown in SEQ ID No. 5, or a heterologous nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 5, wherein said heterologous nucleic acid sequence encodes an enzyme having sucrose permease activity of SEQ ID No. 6; and, optionally, (iii) a heterologous nucleic acid sequence as shown in SEQ ID No. 3, or a heterologous nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 3, wherein said heterologous nucleic acid sequence encodes an enzyme having fructokinase activity of SEQ ID No. 4.
4. The Rhodobacter cell of claim 3, wherein the heterologous nucleic acid sequence as defined in claim 3(i), 3(ii) and 3(iii) is codon-optimized for expression in Rhodobacter.
5. The Rhodobacter cell of claim 3 or 4, wherein the heterologous nucleic acid sequence as defined in claim 3(i), 3(ii) and 3(iii) is under the control of a constitutive promoter.
6. The Rhodobacter cell of claim 5, wherein the constitutive promoter driving the expression of the heterologous nucleic acid sequence encoding the enzyme having invertase activity and the optional heterologous nucleic acid sequence encoding the enzyme having fructokinase activity is the Pppa promoter, preferably comprising a sequence as shown in SEQ ID No. 7, or the PcrtE promoter, and the constitutive promoter driving the expression of the heterologous nucleic acid sequence encoding the enzyme having sucrose permease activity is the Prplm promoter, preferably comprising a sequence as shown in SEQ ID No. 9.
7. The Rhodobacter cell of any one of claims 1 to 6, wherein the Rhodobacter cell exhibits (i) an increased growth rate on sucrose as carbon source, compared to the growth rate on glucose as carbon source, and/or (ii) improved sucrose utilization compared to a wildtype Rhodobacter cell.
8. The Rhodobacter cell of claim 7, wherein sucrose is the sole carbon source and sucrose is refined sucrose, preferably from sugarcane or sugarbeet, less-refined sucrose sugar streams, molasses, thick juice or sugarcane juice.
9. The Rhodobacter cell of any one of claims 1 to 8, further comprising: (i) nucleic acid sequences encoding enzymes of a mevalonate pathway for making isoprenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), (ii) a nucleic acid sequence encoding an enzyme having catalytic activity for the condensation of IPP and DMAPP into geranyl diphosphate (GPP), and (iii) a nucleic acid sequence encoding an enzyme having monoterpene synthase activity in the conversion of GPP into a monoterpene.
10. The Rhodobacter cell of claim 9, wherein the enzyme having monoterpene synthase activity is selected from the group of enzymes having beta-pinene synthase activity, alpha-pinene synthase activity, myrcene synthase activity, limonene synthase activity, linalool synthase activity, sabinene synthase activity, bisabolene synthase activity, and geraniol synthase activity.
11. The Rhodobacter cell of claim 9 or 10, wherein the monoterpene is selected from the group consisting of beta-pinene, myrcene, alpha-pinene, limonene, linalool, sabinene, bisabolene and geraniol.
12. The Rhodobacter cell of any one of claims 1 to 8, further comprising: (i) nucleic acid sequences encoding enzymes of a mevalonate pathway for making isoprenyl pyrophosphate (IPP); (ii) a nucleic acid sequence encoding an enzyme having catalytic activity in the conversion of IPP into farnesyl pyrophosphate (FPP); and (iii) a nucleic acid sequence encoding an enzyme having sesquiterpene synthase activity in the conversion of FPP into a sesquiterpene, wherein the Rhodobacter cell is free of heterologous enzymes having catalytic activity in the reaction of conversion of two molecules of acetyl-CoA to acetoacyl-CoA.
13. The Rhodobacter cell of claim 12, wherein the enzyme having sesquiterpene synthase activity is selected from the group of enzymes having valencene synthase activity, bisabolene synthase activity, bisabolol synthase activity, bergamotene synthase activity, farnesene synthase activity, zizaene synthase activity, santalene synthase activity, zingiberene synthase activity and patchoulol synthase activity.
14. The Rhodobacter cell of claim 12 or 13, wherein the sesquiterpene is valencene, bisabolene, bisabolol, bergamotene, farnesene, zizaene, santalene zingiberene, or patchoulol.
15. The Rhodobacter cell of claim 12, wherein the Rhodobacter cell exhibits an increased production of a sesquiterpene, preferably zingiberene, using sucrose as carbon source, preferably as sole carbon source, compared to the production of said sesquiterpene, preferably zingiberene, using glucose as carbon source, preferably as sole carbon source.
16. The Rhodobacter cell of claim 9 or 12, further comprising: (i) a nucleic acid sequence encoding an enzyme having geranylgeranyl pyrophosphate synthase activity; (ii) a nucleic acid sequence encoding an enzyme having labda-13-en-8-ol diphosphate synthase activity; and (iii) a nucleic acid sequence encoding an enzyme having sclareol synthase activity, wherein the Rhodobacter cell preferably exhibits an increased production of sclareol.
17. Method for preparing a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a flavour, an aroma compound, an UV scavenger, a natural colorant, a natural crop protectant or an isoprenoid moiety containing compound such as coenzyme Q10, comprising culturing a Rhodobacter cell according to any one of the claims 1-16 in a culture medium comprising sucrose as carbon source, preferably sole carbon source, for the monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene, flavour, aroma compound, UV scavenger, natural colorant, natural crop protectant or the isoprenoid moiety containing compound such as coenzyme Q10.
18. Use of the Rhodobacter cell of any one of claims 1 to 16 for the production of a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a flavour, an aroma compound, an UV scavenger, a natural colorant, a natural crop protectant or for production of an isoprenoid moiety containing compound such as coenzyme Q10, preferably utilizing sucrose as carbon source.

Description

The present invention relates to a Rhodobacter cell engineered to utilize sucrose, comprising: (i) a heterologous enzyme having invertase activity; (ii) a heterologous enzyme having sucrose permease activity; and, optionally, (iii) a heterologous enzyme having fructokinase activity, and to a method for producing a Rhodobacter cell engineered to utilize sucrose. The invention further relates to a method for preparing a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a flavour, an aroma compound, an UV scavenger, a natural colorant, a natural crop protectant or an isoprenoid moiety containing compound such as coenzyme Q10, comprising culturing the Rhodobacter cell according to the invention in a culture medium comprising sucrose as carbon source for the monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene, flavour, aroma compound, UV scavenger, natural colorant, natural crop protectant or the isoprenoid moiety containing compound such as coenzyme Q10. In addition, the invention pertains to the use of the Rhodobacter cell of the invention for the production of monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, flavours, aroma compounds, UV scavengers, natural colorants, natural crop protectants or for production of isoprenoid moiety containing compounds such as coenzyme Q10. Carbon source is one of the major cost drivers for industrial production of bulk chemicals from microbial fermentation. Currently, glucose, typically from corn, is the most common carbon source for industrial fermentation in Escherichia coli. Sucrose from sugarcane or sugarbeet, however, would be preferable to corn-based glucose as a carbon substrate for E. coli-based industrial fermentation. First, it is a cheaper substrate as it can be directly fermented, either as cane juice or as molasses, or it can be easily made into pure sugar by high-temperature crystallization, whereas glucose has to be converted from starch by milling and enzymatic hydrolysis. Second, sugarcane sucrose-based bioprocesses are more environmentally friendly and sustainable than glucose-based bioprocesses. This is because bagasse, the fibrous by-product from sugarcane mills, can be utilized to produce energy for the bioprocess, whereas corn glucose-based processes rely on fossil fuels for energy. Finally, as a result of these two primary factors, the associated overall bioprocess cost is decreased relative to that with glucose. In addition, sucrose is highly abundant and readily available. The ability to metabolize sucrose as a carbon source is a highly variable feature among E. coli strains. Sucrose-fermenting strains include the enteropathogenic strains, B-62, EC3132 and its mutants, and E. coli W (Archer C, Kim J, Jeong H, Park JH, Vickers CE, Lee SY, Nielsen LK. 2011. The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics 12:9; V.B. Shukla, S. Zhou, L.P. Yomano, K.T. Shanmugam, J.F. Preston & L.O. Ingram. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26: 689-693, 2004). There are two gene clusters responsible for sucrose catabolism in E. coli: the scr regulon, encoding a sucrose phosphotransferase system (PTS), and the chromosomally carried sucrose catabolism (csc) regulon, encoding a sucrose non-PTS utilization system (Jahreis K, Bentler L, Bockmann J, Hans S, Meyer A, Siepelmeyer J, Lengeler JW. 2002. Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132. J. Bacteriol. 184:5307-5316). Additionally, the E. coli KO11, an ethanologenic derivative of the B strain, has the native ability to ferment sucrose (Moniruzzaman M, Lai X, York SW, Ingram LO (1997) Extracellular melibiose and fructose are intermediates in raffinose catabolism during fermentation to ethanol by engineered enteric bacteria J. Bacteriol. 179: 1880-1886). Genomic DNA from this organism was used to construct a gene library. Transformants of E. coli DH5α were selected for ampicillin resistance and screened for sucrose fermentation using selection plates containing 1% sucrose. Three stable clones growing on sucrose were identified and one was fully sequenced (GenBank accession number AY314757) revealing that the clone contained three complete open reading frames encoding invertase (cscA), and a bicistronic operon (cscKB) encoding fructokinase and an anion symporter for sucrose, respectively. In one study, Bruschi et al. describe the transfer of the csc operon into non-sucrose utilizing E. coli strains (Bruschi M, Boyes SJ, Sugiarto H, Nielsen LK, Vickers CE. 2012. A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains. Biotechnol. Adv. 30:1001-1010). In the study of Shukla et al. (loc. cit.), the sucrose utilization genes (truncated cscR, cscA and cscKB) from the csc regulon from E. coli KO11 were transferred to non-sucrose utilizing E. coli W3110 derivatives, stra