US-12624360-B2 - Microbial respiration of chloroxyanions as a source of oxygen for bioprocessing

Abstract

Aerobic microbial processes comprise culturing microbes comprising a (per)chlorate respiration pathway in a bioreactor in a bioprocess employing microbial respiration of chloroxyanions as a source of oxygen for the bioprocess, in the absence of external addition of molecular oxygen.

Inventors

• John D. Coates

Assignees

• THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date

20260512

Application Date

20230507

Claims (1)

1. 1 . A method of aerobic microbial processing comprising: culturing microbes comprising a (per)chlorate respiration pathway in a bioreactor in a bioprocess employing microbial respiration of chloroxyanions as a source of oxygen for the bioprocess, in the absence of external addition of molecular oxygen, wherein the microbes are genetically engineered to comprise an operative microbial (per)chlorate respiration pathway encoding: (i) a perchlorate reductase which effects reduction of (per)chlorate to chlorite (ClO 2 − ); (ii) a chlorite dismutase which effects dismutation of the chlorite into chloride and molecular oxygen (O 2 ); and (iii) a cytochrome oxidase which effects reduction of the molecular oxygen; wherein the pathway enables the engineered microbe to grow in the bioreactor with (per)chlorate as its sole electron acceptor in the absence of oxygen, wherein the microbes are obligately aerobic methanotrophs, that are Methylocystis trichosporium, and the bioprocess converts methane (CH 4 ) into polyhydroxybutyrate (PHB).

Description

The bioeconomy has estimated an annual value of $960B or 5% of the 2016 US GDP. The bioeconomy was also recently recognized in a US National Academy of Science report as a burgeoning field of opportunity, both to ameliorate climate change and to move away from petrochemicals towards a sustainable future. While the bioeconomy is based on any process that involves the sustainable conversion of biological material into products, i.e. bio-based chemicals, fuels, plastics, building materials, etc., biotechnology is the purported primary mode manufacturing. The majority of industrial microbial processes are based on aerobic metabolisms because of the favorable biochemical energetics and metabolic versatility of aerobic microorganisms. However, these processes are conversely the most energy intensive due to the extensive agitation required to dissolve poorly soluble oxygen into an aqueous system. While anaerobic microbial processes are preferential due to the significant cost savings, many microbial transformations require molecular oxygen as a co-substrate. These costs can be as much as 50% of the capex and 20% of operational costs. Furthermore, the energy required is often sourced from fossil fuels significantly offsetting the environmental benefits of the sustainable process. Quite often, it is the burden of these costs that prevent biotechnological processes from competing successfully with existing petrochemical manufacturing, especially for commodity chemicals. For example, while there are several organisms known that can produce bioplastics from sustainable feedstocks, these are prevented from successfully penetrating the market because of the additional cost premium when compared to petrochemical plastics. We previously disclosed Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1, Iain C. Clark, et al.,2015, mBio 6(3) e00282-15: and (Per)Chlorate-Reducing Bacteria Can Utilize Aerobic and Anaerobic Pathways of Aromatic Degradation with (Per)Chlorate as an Electron Acceptor, Charlotte I. Carlström, et al., 2015, mBio 6 (3) e02287-14. These basic research projects provided further biochemical and genetic insight into the respiratory (per)chlorate reduction pathway and identification of components. The present invention takes advantage of these findings, as well as our research showing that (per)chlorate can be metabolized to provide oxygen under anaerobic conditions to allow for oxygen dependent metabolisms, to provide novel bioreactors and bioprocesses. The invention overcomes prior limitations and allows for aerobic microbial processes in the absence of the external addition of molecular oxygen or the need for energy-intensive agitation required for oxygen dissolution. SUMMARY OF THE INVENTION The invention provides novel bioreactors and bioprocesses employing microbial respiration of chloroxyanions as a source of oxygen for industrial-scale bioprocessing productions. In an aspect, the invention provides a method of aerobic microbial processing comprising: culturing microbes comprising a (per)chlorate respiration pathway in a bioreactor in a bioprocess employing microbial respiration of chloroxyanions as a source of oxygen for the bioprocess, in the absence of external addition of molecular oxygen. In embodiments: the bioprocess comprises production of drugs, commercial or industrial enzymes, bioplastics or bioplastic precursors, biofuels or biofuel precursors, commodity chemicals, cosmetics, foods, such as plant-protein based meat substitutes, or food additives, such as citric acid;the microbes are obligately aerobic methanotrophic, and the bioprocessing converts the greenhouse gas methane (CH4) into the biopolymer polyhydroxybutyrate (PHB), polyhydroxyalkanoate (PHA), or polylactic acid (PLA);the microbes are obligately aerobic eukaryotic fungi, and the bioprocess utilizes complex lignin-celluosic feedstocks;the microbial respiration comprises a pathway comprising: (i) reduction of (per)chlorate to chlorite (ClO2−) by perchlorate reductase; (ii) dismutation of chlorite into chloride and molecular oxygen (O2) by chlorite dismutase; and (iii) reduction of molecular oxygen by cytochrome oxidase;the microbial respiration comprises a pathway comprising: (i) reduction of chlorate to chlorite (ClO2−) by a protein belonging to the DMSO reductase superfamily of molybdopterin oxidoreductases; (ii) dismutation of chlorite into chloride and molecular oxygen (O2) by chlorite dismutase; and (iii) reduction of molecular oxygen by cytochrome oxidase;the microbial (per)chlorate respiration pathway is engineered;the microbes are genetically engineered to comprise an operative microbial (per)chlorate respiration pathway encoding: (i) a perchlorate reductase which effects reduction of (per)chlorate to chlorite (ClO2−); (ii) a chlorite dismutase which effects dismutation of the chlorite into chloride and molecular oxygen (O2); and (iii) a cytochrome oxidase which effects reduction of the