De novo biosynthesis of 3-hydroxy-3-methylbutyrate as anti-catabolic supplement by metabolically engineered Escherichia coli.

3-Hydroxy-3-methylbutyrate (HMB) is a five-carbon branch-chain hydroxy acid currently used as a dietary supplement to treat sarcopenia and exercise training. However, its current production relies on conventional chemical processes which require toxic substances and are generally non-sustainable. While bio-based syntheses of HMB have been developed, they are dependent on biotransformation of its direct precursors which are generally costly. Therefore, in this work, we developed a synthetic de novo HMB biosynthetic pathway that enables HMB production from renewable resources. This novel HMB biosynthesis employs heterologous enzymes from mevalonate pathway and myxobacterial iso-fatty acid pathway for converting acetyl-CoA to HMB-CoA. Subsequently, HMB-CoA is hydrolyzed by a thioesterase to yield HMB. Upon expression of this pathway, our initial Escherichia coli strain produced 660 mg/L of HMB from glucose in 48 hours. Through optimization of coenzyme A removal from HMB-CoA and genetic operon structure, our final strain achieved HMB production titer of 17.7 g/L in glucose minimal media using a bench-top bioreactor. This engineered strain was further demonstrated to produce HMB from other renewable carbon sources such as xylose, glycerol, and acetate. The results from this work provided a flexible and environmentally benign method for producing HMB.

CRISPRi-enhanced direct photosynthetic conversion of carbon dioxide to succinic acid by metabolically engineered cyanobacteria.

Engineering photoautotrophic microorganisms to directly convert carbon dioxide into platform chemicals is an attractive approach for chemical sustainability and carbon mitigation. Here, an engineered cyanobacterium Synechococcus elongatus PCC 7942 was developed to produce succinic acid directly from ambient carbon dioxide. Inhibition of succinate dehydrogenase and glycogen synthase by CRIPSR interference increased carbon flux towards succinic acid. Dual inhibition of these two genes led to an 82 % increase in titer. The resulting strain produced 4.8 g/L of succinic acid in a 28-days cultivation. However, cells after the 28-days cultivation became non-viable and cannot continue production. This issue was addressed by re-inoculation with fresh cells into the production medium. This strategy enabled continuous succinic acid accumulation, reaching a final titer of 8.9 g/L. This study provides a sustainable route to succinic acid directly from carbon dioxide and a potential method to overcome the low titer limitation of cyanobacterial-based bioproduction for practical applications.

Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria.

Direct conversion of carbon dioxide into chemicals using engineered autotrophic microorganisms offers a potential solution for both sustainability and carbon mitigation. Butyrate is an important chemical used in various industries, including fragrance, food, and plastics. A model cyanobacterium Synechococcus elongatus PCC 7942 was engineered for the direct photosynthetic conversion of CO 2 to butyrate. An engineered Clostridium Coenzyme A (CoA)-dependent pathway leading to the synthesis of butyryl-CoA, the precursor to butyrate, was introduced into S. elongatus PCC 7942. Two CoA removal strategies were then individually coupled to the modified CoA-dependent pathway to yield butyrate production. Similar results were observed between the two CoA removal strategies. The best butyrate producing strain of S. elongatus resulted in an observed butyrate titer of 750 mg/L and a cumulative titer of 1.1 g/L. These results demonstrated the feasibility of photosynthetic butyrate production and expanded the chemical repertoire accessible for production by photoautotrophs.