Heterologous production of α-Carotene in Corynebacterium glutamicum using a multi-copy chromosomal integration method.

The carotenoid, α-carotene, is very beneficial for human health and wellness, but microbial production of this compound is notoriously difficult, due to the asymmetric rings on either end of its terpenoid backbone. Here, we report for the first time the efficient production of α-carotene in the industrial bacterium Corynebaterium glutamicum by using a combined pathway engineering approach including evaluation of the performance of different cyclases and analysis of key metabolic intermediates to determine flux bottlenecks in the carotenoid biosynthesis pathway. A multi-copy chromosomal integration method was pivotal in achieving stable expression of the cyclases. In fed-batch fermentation, 1,054 mg/L of α-carotene was produced by the best strain, which is the highest reported titer achieved in microbial fermentation. The success of increased α-carotene production suggests that the multi-copy chromosomal integration method can be a useful metabolic engineering tool for overexpression of key enzymes in C. glutamicum and other bacterium as well.

Model-based control for column-based continuous viral inactivation of biopharmaceuticals.

Batch low-pH hold is a common processing step to inactivate enveloped viruses for biologics derived from mammalian sources. Increased interest in the transition of biopharmaceutical manufacturing from batch to continuous operation resulted in numerous attempts to adapt batch low-pH hold to continuous processing. However, control challenges with operating this system have not been directly addressed. This article describes a low-cost, column-based continuous viral inactivation system constructed with off-the-shelf components. Model-based, reaction-invariant pH controller is implemented to account for the nonlinearities with Bayesian estimation addressing variations in the operation. The residence time distribution is modeled as a plug flow reactor with axial dispersion in series with a continuously stirred tank reactor, and is periodically estimated during operation through inverse tracer experiments. The estimated residence time distribution quantifies the minimum residence time, which is used to adjust feed flow rates. Controller validation experiments demonstrate that pH and minimum residence time setpoint tracking and disturbance rejection are achieved with fast and accurate response and no instability. Viral inactivation testing demonstrates tight control of logarithmic reduction values over extended operation. This study provides tools for the design and operation of continuous viral inactivation systems in service of increasing productivity, improving product quality, and enhancing patient safety.

Yeast-Based Synthetic Biology Platform for Antimicrobial Peptide Production.

Antibiotic resistance is one of the most challenging global health threats in our society. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics for the treatment of drug-resistant infections. However, they are limited by their high manufacturing cost. Engineering living organisms represents a promising approach to produce such molecules in an inexpensive manner. Here, we genetically modified the yeast Pichia pastoris to produce the prototypical AMP apidaecin Ia using a fusion protein approach that leverages the beneficial properties ( e.g., stability) of human serum albumin. The peptide was successfully isolated from the fusion protein construct, purified, and demonstrated to have bioactivity against Escherichia coli. To demonstrate this approach as a manufacturing solution to AMPs, we scaled-up production in bioreactors to generate high AMP yields. We envision that this system could lead to improved AMP biomanufacturing platforms.